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Method for selective conjugation of analytes to enzymes without unwanted enzyme-enzyme cross-linking

an enzyme and analyte technology, applied in the field of analyte reagents, can solve the problems of undesirable or inconvenient assays of analyte-conjugated analytes, small amount of conjugate can be purified, etc., and achieve the effect of eliminating unwanted cross-linking of enzymes and simple and efficient conjugation

Inactive Publication Date: 2005-02-10
BOARD OF RGT NEVADA SYST OF HIGHER EDUCATION ON BEHALF OF THE UNIV OF NEVADA RENO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006] The present invention is a simple and efficient method of conjugating an analyte to an enzyme that substantially eliminates the cross-linking of the enzyme by employing a pretreatment step of blocking surface accessible carboxyl moieties in the enzyme peptide prior to the conjugation reaction.
[0007] The method involves treating an enzyme containing free, surface-accessible carboxyl moieties with a blocking agent such that the free carboxyl moieties become non-reactive prior to the conjugation reaction with the desired analyte. Since cross-linking of the blocked enzymes is minimized or prevented, the yield of the analyte-enzyme conjugate formation and the purity of the conjugates formed are significantly improved compared to prior art methods.
[0008] As exemplified herein, the enzymes modified (i.e., blocked) according to the invention preferably exhibit no significant decrease in catalytic properties. A significant decrease in catalytic properties of an enzyme on modification is one in which one or more measurable indicators of catalytic activity of the modified (blocked) enzyme are decreased more than about 50% compared to the non-modified (non-blocked) enzyme by the modification employed. Therefore, the invention can be applied to improve the preparation of any analyte-enzyme conjugate of any analyte where the enzyme contains surface accessible carboxyl moieties that may trigger self aggregation of the enzyme molecules and thus reduce efficiency of analyte-enzyme conjugate formation.
[0010] Preferred coupling reagents and blocking groups are at least partially water-soluble for improved efficiency of reaction. Further, the coupling reagent, such as a carbodiimide, can be bound to a solid support, such as a polydextran or agarose polymers. Carbodiimides, particularly those that are at least partially water-solub le are preferred coupling reagents. Carbodiimides useful in the methods of this invention, include, but are not limited to, 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide methiodide (and other forms of EDC that are water-soluble) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-methyl-p-toluenesulfonate. In a specific embodiment, the use of a carbodiimide (or other coupling reagent) carrying at least one bulky group is preferred for use in blocking enzymes in which one or more carboxyl groups may be associated with the enzyme active site. In another specific embodiment, the use of a carbodiimide (or other coupling reagent) bound to a solid support or polymer group is preferred for use in blocking enzymes in which one or more carboxyl groups may be associated with the enzyme active site. Carbodiimides having at least one bulky group include those that contain a cyclic alkyl or cyclic heteroakyl group, such as a cyclohexyl group, or a morpholino group.

Problems solved by technology

Although there have been continuous efforts to improve various aspects of the assays that are commonly used in clinical medicine and research laboratories, there still exists a significant problem of preparing a large quantity of an analyte-enzyme conjugate of high purity by a simple and efficient process.
Therefore, it is necessary to carry out laborious chromatographic purification steps to obtain the analyte-enzyme conjugates separated from cross-linked materials and as a result only a small amount of the conjugate can be purified at a time.
Those of ordinary skill in the art would not expect, in general, that art known blocking methods could be employed in forming analyte-enzyme conjugates without loss of significant enzyme activity which would render the analyte-conjugates undesirable or not useful in assays.

Method used

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  • Method for selective conjugation of analytes to enzymes without unwanted enzyme-enzyme cross-linking
  • Method for selective conjugation of analytes to enzymes without unwanted enzyme-enzyme cross-linking
  • Method for selective conjugation of analytes to enzymes without unwanted enzyme-enzyme cross-linking

Examples

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example 1

Enzyme Modification

[0042] In order to block free carboxyl moieties on the horseradish peroxidase (HRP) enzyme, 4.5 mg of HRP (Biozyme Laboratories, San Diego, Calif. was dissolved in 1 ml of a mixture of 0.1M (2-[N-morpholino]ethanesulfonic acid) (used as a buffer) and 0.1M Tris (hydroxymethyl) aminomethane (a large excess of amine blocking agent) in purified dionized water that had been titrated to pH 4.75. Thereafter 15 mg of 1-[3-dimethyl-amino)propyl]-3-ethylcarbodiimide methiodide was added and the mixture was reacted for 2 hours at room temperature while stirring. Upon completion of the reaction, the mixture was dialyzed against 2 liters of PBS pH 7.4 overnight at 4° C. The modified enzyme was then removed from dialysis and assayed for activity using TMB (3,3′,5,5′-tetramethylbenzidine) peroxidase substrate [Porstman and Kiessing (1992) J. Immunol. Methods 150:521]. The details of the coupling reaction can be found in Grabarek, Z. et al. (1990) Anal. Biochem. 185:244-248; Wil...

example 2

cAMP Assay

[0044] The following is an example of the use of an enzyme-conjugate prepared according to the invention in a cAMP Assay. [Horton et al. (1992) J. Immunological Meth. 15:31-40; Steiner et al. (1969) Proc. Natl. Acad. Sci. 64:367-373].

[0045] Affinity purified goat-anti-rabbit Fc antibody (27.5 μl) was added to 10 ml of phosphate buffered saline (PBS) (PBS is 0.15M NaCl, 0.01M sodium phosphate in purified water at pH 7.4) and made homogeneous. This solution (90 μl) was added to each well of a Costar High Binding EIA microtiter plate. The plate was then incubated for 4 hours at room temperature. The contents of the plate were discarded and 350 μl of a 3% solution of normal goat serum in PBS was added to each well. The plate was incubated at room temperature for 1 hour and the contents of the plate were discarded. Each well was then washed with 150 μl of washing buffer (washing buffer is 0.05% Tween 20 detergent in PBS). Thereafter 75 μl of a 1:10000 solution of affinity pur...

example 5

Preparation of Succinyl-cAMP-RP Conjugate

[0047] Succinyl-cAMP was conjugated to blocked horseradish peroxidase prepared as described above, using 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS).

[0048] Approx. 1.366 mg of 2-O-monosuccinyl-cAMP and approx. 3.5 mg of N-hydroxysulfosuccinimide were added to the above horseradish peroxidase solution (˜4.6 mg in 1.5 ml PBS, pH 7.4). Approximately 13 mg of 1-[3-dimethylamino)propyl]-3-ethylcarbodiimide methiodide was then added and the solution was mixed on an Orbital shaker overnight at 4° C. The modified enzyme was dialyzed against 1 liter of PBS overnight four times. Thimerosal (0.01%) was added to the enzyme conjugate and stored at 4° C.

[0049] 2-O-monosuccinyl-cGMP can be coupled to the HRP in a similar manner.

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Abstract

The present invention provides a simple and efficient method of preparing an analyte-enzyme conjugate where the enzyme contains free, surface-accessible carboxyl moieties without generating undesired, cross-linked enzymes, while preserving CN the functionality of the enzyme. The method involves treating an enzyme with a blocking agent such that the free carboxyl moieties become non-reactive prior to the conjugation reaction with the desired analyte. The yield of the analyte-enzyme conjugate and the purity of the conjugates formed are high since cross-linking of the blocked enzymes is minimized or prevented. The invention is generally useful in preparing any conjugates of an analyte of interest and an enzyme containing surface accessible carboxyl moieties. The invention is particularly useful in preparing conjugates of any analyte of interest and horseradish peroxidase, alkaline phos-phatase or acetylcholine esterase. The conjugates prepared according to the invention are useful in a variety of assays including but not limited to enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and the like. The invention is further directed to analyte-enzyme conjugates prepared by the inventive method and to kits which contain an analyte-enzyme conjugate prepared by the methods herein for the detection and / or quantitation of an analyte in a sample.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority from U.S. Provisional Patent Application No. 60 / 339,842, filed Nov. 16, 2001, which is incorporated herein in its entirety.FIELD OF THE INVENTION [0002] The present invention relates to analytical reagents and methods of making such reagents, and particularly to improved processes of selectively preparing a conjugate of an analyte-enzyme without generating undesired multimers of the enzyme via cross-linking. The invention also relates to conjugates prepared by the processes. BACKGROUND OF THE INVENTION [0003] Although there have been continuous efforts to improve various aspects of the assays that are commonly used in clinical medicine and research laboratories, there still exists a significant problem of preparing a large quantity of an analyte-enzyme conjugate of high purity by a simple and efficient process. For example, in a typical enzyme-linked immunosorbent assay (ELISA), given analyte-enzyme conj...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/00C12Q1/48G01N33/535
CPCC12N9/96G01N33/535
Inventor LOMBARDI, VINCENT CSCHOOLEY, DAVID A.
Owner BOARD OF RGT NEVADA SYST OF HIGHER EDUCATION ON BEHALF OF THE UNIV OF NEVADA RENO